Chapter 1

Introduction to free radicals, antioxidants and brief overview of

oximes

Destiny is not a matter of chance, it is a matter of choice;

It is not a thing to be waited for, it is a thing to be achieved.

-William Jennings Bryan

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1.1 Free radicals

Nutrients that are commonly used by animal and plant cells in respiration

include sugar, amino acids, fatty acids and a common oxidizing agent (electron

acceptor) is molecular oxygen (O2). The energy stored in ATP can then be used to drive

processes requiring energy, including biosynthesis, locomotion or transportation of

molecules across cell membranes. The ability to utilize oxygen has provided humans

with the benefit of metabolizing nutrients. Oxygen is a highly reactive atom that is

capable of becoming part of potentially damaging molecules commonly called “free

radicals.”

Free radicals can be defined as molecules or molecular fragments containing

one or more unpaired electrons in atomic or molecular orbitals.1 Free radicals may have

positive, negative or zero charge, i.e., they have an unpaired electron on an open shell

configuration, which causes them to seek out and capture electrons from other

substances in order to neutralize themselves. Although the initial attack causes the free

radical to become neutralized, another free radical is formed in the process, causing a

chain reaction to occur and until subsequent free radicals are deactivated, thousands of

free radical reactions can occur within seconds of the initial reaction.

The formation of radicals may involve breaking of covalent bonds

homolytically, radicals requiring more energy to form are less stable than those

requiring less energy. Homolytic bond cleavage most often happens between two atoms

of similar electronegativity. In organic chemistry this is often the O-O bond or O-N

bonds. Radical ions do exist, most species are electrically neutral. Radicals may also be

formed by single electron oxidation or reduction of an atom or molecule. An example is

the production of superoxide by the electron transport chain. Free radicals are capable

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of attacking the healthy cells of the body, causing them to lose their structure and

function. Cell damage caused by free radicals appears to be a major contributor to

aging and to degenerative diseases of aging such as cancer, cardiovascular disease,

cataracts, immune system decline and brain dysfunction. Overall, free radicals have

been implicated in the pathogenesis of at least 50 diseases.2, 3

Reactive oxygen species

Reactive oxygen species (ROS) is a term which encompasses all highly

reactive, oxygen-containing molecules, including free radicals. These include oxygen in

its triplet state (3O2) or singlet state (1O2), superoxide anion (O2⁻), hydroxyl radical

(·OH), nitric oxide (NO), peroxynitrite (ONOO⁻), hypochlorous acid (HOCl), hydrogen

peroxide (H2O2), alkoxyl radical (RO·) and the peroxyl radical (RO·2).

Other free radicals are carbon-centered free radical (CCl3·) that arises from the

attack of an oxidizing radical on an organic molecule. Hydrogen centered radicals

result from attack of the H atom (H). Another form is the sulphur-centered radical

produced in the oxidation of glutathione resulting in the thioyl radical (R-S·). A

nitrogen-centered radical also exists, for example the phenyl diazine radical.

All are capable of reacting with membrane lipids, nucleic acids, proteins and

enzymes and other small molecules, resulting in cellular damage. ROS form as a

natural byproduct of the normal metabolism of oxygen and have important roles in cell

signaling. However, during times of environmental stress (e.g. UV or heat exposure)

ROS levels can increase drastically, which can result in significant damage to cell

structures. This cumulates into a situation known as oxidative stress.

ROS are also generated by exogenous sources such as ionizing

radiation. Exogenous ROS can be produced from pollutants, food additives, tobacco,

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smoke, drugs, xenobiotics and modern day lifestyles related stress is also a contributing

factor to excess free radicals circulating in our body. Consequently, things like vigorous

exercise, which accelerates cellular metabolism; chronic inflammation, infections and

other illnesses; exposure to allergens and the presence of “leaky gut” syndrome; and

exposure to drugs or toxins such as cigarette smoke, pollution, pesticides and

insecticides may all contribute to an increase in the body’s oxidant load.

Concept of oxidative stress

The term is used to describe the condition of oxidative damage resulting when

the critical balance between free radical generation and antioxidant defenses is

unfavorable.4 Oxidative stress is associated with damage to a wide range of molecular

species including lipids, proteins and nucleic acids.5 Short-term oxidative stress may

occur in tissues injured by trauma, infection, heat injury, hypertoxia, toxins and

excessive exercise. These injured tissues produce increased radical generating enzymes

(e.g., xanthine oxidase, lipogenase, cyclooxygenase) activation of phagocytes, release

of free iron, copper ions or a disruption of the electron transport chains of oxidative

phosphorylation, producing excess ROS. The initiation, promotion and progression of

cancer, as well as the side-effects of radiation and chemotherapy, have been linked to

the imbalance between ROS and the antioxidant defense system. ROS have been

implicated in the induction and complications of diabetes mellitus, age-related eye

disease and neurodegenerative diseases such as Parkinson's disease.6

Oxidative stress and human diseases

A role of oxidative stress has been postulated in many conditions, including

atherosclerosis, inflammatory condition, certain cancers and the process of aging.

Oxidative stress is now thought to make a significant contribution to all inflammatory

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diseases (arthritis, vasculitis, glomerulonephritis, lupus erythematous, adult respiratory

diseases syndrome), ischemic diseases (heart diseases, stroke, intestinal ischema),

hemochromatosis, acquired immunodeficiency syndrome, emphysema, organ

transplantation, gastric ulcers, hypertension and preeclampsia, neurological disorder

(alzheimer's disease, parkinson's disease, muscular dystrophy), alcoholism, smoking-

related diseases and many others.7

Oxidative damage to DNA

At high concentrations, ROS can be important mediators of damage to cell

structures, nucleic acids, lipids and proteins.8 The hydroxyl radical is known to react

with all components of the DNA molecule, damaging both the purine and pyrimidine

bases and also the deoxyribose backbone. The most extensively studied DNA lesion is

the formation of 8-OH-G. Permanent modification of genetic material resulting from

these “oxidative damage” incidents represents the first step involved in mutagenesis,

carcinogenesis and ageing.

Figure 1.1. Oxidative damage to DNA by ROS

Figure 1.1. shows the schematic representation of singlet molecular oxygen

generation (1O2) by methylene blue photosensitization (type II) or endoperoxide (DHP-

NO2) thermolysis and DNA oxidation by 1O2 leading to the formation of base

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oxidation, as for example 8-oxoG, that is recognised by farmamido pyramidine DNA

glycolase (FPG) enzyme that cleaves DNA in the site of the lesion.

Mitochondria are unique organelles, as they are the main site of oxygen

metabolism, accounting for approximately 85–90% of the oxygen consumed by the

cell. Incomplete processing of oxygen and /or release of free electrons results in the

production of oxygen radicals. Mitochondria constantly metabolize oxygen thereby

producing ROS as a byproduct. These organelles have their own ROS scavenging

mechanisms that are required for cell survival. It has been shown, however, that

mitochondria produce ROS at a rate higher than their scavenging capacity, resulting in

the incomplete metabolism of approximately 1–3% of the consumed oxygen.

The byproducts of incomplete oxygen metabolism are superoxide (O2−), hydrogen

peroxide (H2O2), and hydroxyl radical (·OH). The formation of superoxide occurs via

the transfer of a free electron to molecular oxygen. This reaction occurs at specific sites

of the electron transport chain (ETC), which resides in the inner

mitochondrial membrane (Figure 1.2). ETC complexes I (NADH dehydrogenase) and

III (ubisemiquinone) produce most of the superoxide, which is then scavenged by the

mitochondrial enzyme manganese superoxide dismutase (MnSOD) to produce H2O2.

Figure 1.2. Generation of ROS in electron transport chain

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Lipid peroxidation

Lipid peroxidation is a free radical process involving a source of secondary free

radical, which further can act as second messenger or can directly react with other

biomolecule, enhancing biochemical lesions. Lipid peroxidation occurs on poly

saturated fatty acid located on the cell membranes and it further proceeds with radical

chain reaction. Hydroxyl radical is thought to initiate ROS and remove hydrogen atom,

thus producing lipid radical and further converted into diene conjugate. Further, by

addition of oxygen it forms peroxyl radical; this highly reactive radical attacks another

fatty acid forming lipid hydroperoxide (LOOH) and a new radical. Thus, lipid

peroxidation is propagated. Due to lipid peroxidation, a number of compounds are

formed, for example, alkanes, malanoaldehyde and isoprotanes. These compounds are

used as markers in lipid peroxidation assay and have been verified in many diseases

such as neurogenerative diseases, ischemic reperfusion injury and diabetes.9

Oxidative damage to protein

Proteins can be oxidatively modified in three ways: oxidative modification of

specific amino acid, free radical mediated peptide cleavage and formation of protein

cross-linkage due to reaction with lipid peroxidation products. Protein containing

amino acids such as methionine, cystein, arginine, and histidine seem to be the most

vulnerable to oxidation.10 Free radical mediated protein modification increases

susceptibility to enzyme proteolysis. Oxidative damage to protein products may affect

the activity of enzymes, receptors and membrane transport. Oxidatively damaged

protein products may contain very reactive groups that may contribute to damage to

membrane and many cellular functions. Peroxyl radical is usually considered to be free

radical species for the oxidation of proteins. ROS can damage proteins and produce

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carbonyls and other amino acids modification including formation of methionine

sulfoxide and protein carbonyls and other amino acids modification including

formation of methionine sulfoxide and protein peroxide.

Figure 1.3. Protein oxidation by ROS

Protein oxidation (Figure 1.3) affects the alteration of signal transduction

mechanism, enzyme activity, heat stability and proteolysis susceptibility, which leads

to aging.

1.2. Antioxidant

Antioxidant means "against oxidation. Antioxidants are effective because they

are willing to give up their own electrons to free radicals. When a free radical gains the

electron from an antioxidant it no longer needs to attack the cell and the chain reaction

of oxidation is broken. After donating an electron an antioxidant becomes a free radical

by definition. Antioxidants in this state are not harmful because they have the ability to

accommodate the change in electrons without becoming reactive.11 Antioxidants are

capable of stabilizing or deactivating, free radicals before they attack cells.

Antioxidants are absolutely critical for maintaining optimal cellular and systemic health

and well-being.

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The chemistry of antioxidants

It involves the mechanism of action of antioxidant. Two principle mechanisms

of action have been proposed for antioxidants. The first is a chain-breaking mechanism

by which the primary antioxidants donate electrons to the free radicals present in the

system, example lipid radicals.

The second mechanism involves removal of ROS and RNS (reactive nitrogen species)

initiator by quenching chain initiator catalyst.

Chain reactions of free radicals

Initiation stage

(i) RH R˙+ H˙

(ii) 2R˙ + O2 2ROO˙

(iii) 2ROOH ROO˙ + RO˙ + H2O

Propagation stage

(iv) R˙ + O2 ROO˙

(v) ROO˙ + RH ROOH + R˙

(vi) RO˙ + RH ROH + R˙

Termination stage

(vii) R˙ + R˙ R – R

(viii) R˙ + ROO˙ ROOR

(ix) ROO˙ + ROO˙ ROOR + O2

(x) Antioxidants + O2 oxidized antioxidants

Further, in free radical chain reactions, when fats are in contact with oxygen, it

forms unsaturated fatty acids which give rise to free radicals. Also hydro peroxide

which exist in trace quantities prior to oxidation reaction, break down to yield radicals

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which abstract hydrogen atom from another molecule and become a hydroperoxide

producing further radicals. The antioxidants added to it, will neutralize the free radicals

by donating one of their own electrons ending the reactions. These occur generally in

the body.

Types of antioxidants

Antioxidants have been classified based on their occurrence, mode of action,

kinetics and solubility.

I) Based on their occurrence

There are two types (a) Natural antioxidants (b) Synthetic antioxidants

a) Natural antioxidants

They are the chain breaking antioxidants which react with radicals and convert

them into more stable products. Antioxidants of this group are mainly phenolic in

structures and include the following12:

i) Antioxidants minerals - These are co factor of antioxidants enzymes. Their absence

will definitely affect metabolism of many macromolecules such as carbohydrates.

Examples include selenium, copper, iron, zinc and manganese.

ii) Antioxidants vitamins – These are needed for most body metabolic functions

includes -vitamin C, vitamin E, vitamin B.

iii) Phytochemicals - These are phenolic compounds that are neither vitamins nor

minerals. These includes: Flavonoids, catechins, carotenoids, beta carotene,

lycopene, herbs and spices-source include diterpene, rosmariquinone, thyme,

nutmeg, clove, black pepper, ginger, garlic and curcumin and derivatives.

b) Synthetic antioxidants

These are phenolic compounds that perform the function of capturing free

radicals and stopping the chain reactions, the compounds includes: butylated hydroxyl

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anisole (BHA), butylated hydroxyl toluene (BHT), propyl gallate (PG), metal chelating

agent (EDTA), tertiary butyl hydroquinone (TBHQ) and nordihydro guaretic acid

(NDGA).

II) Based on their mode of action

There are two types (a) Primary antioxidants (b) Secondary antioxidants

a) Primary antioxidants:

These interrupt the primary oxidation cycle by removing the propagating

radicals. Such compounds are also called chain breaking antioxidants. They have

reactive OH or NH groups (Hindered phenols and secondary aromatic amines).

Inhibition occurs via a transfer of a proton to the free radical species. The resulting

radical is stable and does not abstract a proton from the polymer chain.

Examples: BHA, BHT, TBHQ, PG, NDGA, hydroquinone, auxomers, olive oil,

carotenoids, steroids, ethoxy quins.

b) Secondary antioxidants:

These compounds are also called preventative antioxidants as they interrupt the

oxidative cycle by preventing or inhibiting the formation of free radicals. Phosphites or

phosphonites, organic sulphur containing compounds and dithiophosphonates are

widely used to achieve this, acting as peroxide decomposers. Secondary antioxidants

frequently referred to as hydroperoxide decomposers; decompose hydroperoxides into

non-radical, non-reactive and thermally stable products.

Examples: Thiodipropioncacid, ascorbic acid, dilauryl, sulphates, erythorbic acid,

polyphosphates, EDTA, phytic acid, lecithin, distearyl ester, nitrates, amino acids,

flavanoids, β-carotene, tea extracts, zinc, selenium, vitamin-C, spice, tartaric acid.

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There are several enzyme systems that catalyze reactions to neutralize free

radicals and reactive oxygen species. These form the body’s endogenous defense

mechanisms to help protect against free radical-induced cell damage. The antioxidant

metabolizes oxidative toxic intermediates. These enzymes include:

Glutathione enzymes and system: Glutathione, an important water-soluble antioxidant,

is synthesized from the amino acids glycine, glutamate and cysteine. Glutathione can

directly neutralize ROS such as lipid peroxides, and also plays a major role in

xenobiotic metabolism. The glutathione system includes glutathione, glutathione

reductase, glutathione peroxidases and glutathione ''S''-transferases.

Lipoic acid: This is another important endogenous antioxidant. It is categorized as

“thiol” or “biothiol”. These are sulfur-containing molecules that catalyze the oxidative

decarboxylation of alpha-keto acids, such as pyruvate and alphaketoglutarate, in the

Krebs cycle. Lipoic acid and its reduced form, dihydrolipoic acid (DHLA), neutralize

the free radicals in both lipid and aqueous domains and as such has been called a

“universal antioxidant.”

Superoxide dismutase: Superoxide dismutase (SOD) is a class of enzymes that catalyse

the breakdown of the superoxide anion into oxygen and hydrogen peroxide. These

enzymes are present in almost all aerobic cells and in extracellular fluids.

Catalases: Catalases are enzymes that catalyze the conversion of hydrogen peroxide to

water and oxygen, using either an iron or manganese cofactor. This is found in

peroxisomes in most eukaryotic cells. Its only substrate is hydrogen peroxide. It follows

a ping-pong mechanism. Here, its cofactor is oxidized by one molecule of hydrogen

peroxide and then regenerated by transferring the bound oxygen to a second molecule

of substrate.

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III) Kinetically antioxidants can be classified into six categories as below:

(a) Antioxidants that break chains by reacting with peroxyl radicals having weak O-H

or N-H bonds; phenol, napthol, hydroquinone, aromatic amines and aminophenols.

(b) Antioxidants that break chains by reacting with alkyl radicals: quinones, nitrones,

iminoquinones.

(c) Hydro peroxide decomposing antioxidants; sulphide, phosphide, thiophosphate.

(d) Metal deactivating antioxidants: diamines, hydroxyl acids and bifunctional

compounds.

(e) Cyclic chain termination by antioxidants: aromatic amines, nitroxyl radical,

variable valence metal compounds.

(f) Synergism of action of several antioxidants: phenol sulphide in which phenolic

group reacts with peroxyl radical and sulphide group with hydro peroxide.

IV) Based on their solubility

Antioxidants are classified into two broad divisions

a) Water soluble antioxidants

In general, water-soluble antioxidants react with oxidants in the cell cytosol and

the blood plasma. The water soluble antioxidants include vitamin-C, glutathione

peroxidase, superoxide dismutase, and catalase.13

b) Lipid soluble antioxidants

While lipid-soluble antioxidants protect cell membranes from lipid

peroxidation. These compounds may be synthesized in the body or obtained from the

diet.14 The lipid soluble antioxidants are vitamin-E, beta-carotene, and coenzyme Q.15

Of these, vitamin-E is considered the most potent chain breaking antioxidant within the

membrane of the cell.

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1.3. Epidemiology of antioxidants

Investigators conducting epidemiologic studies of antioxidant nutrients typically

estimate intake of the nutrient from foods and supplements using questionnaire or food-

record techniques and/or rely upon a biochemical measure of exposure to the nutrient

of interest.16

Moreover oxidised LDLs show cytotoxic potential which is probably

responsible for endothelial cell damage and macrophage degeneration in the

atherosclerotic human plaque. Following the oxidation hypothesis of atherosclerosis the

role of natural antioxidants, i.e. vitamin-C, vitamin-E and carotenoids, has been

investigated in a large number of epidemiological, clinical and experimental studies.

Animal studies indicate that dietary antioxidants may reduce atherosclerosis

progression, and observational data in humans suggest that antioxidant vitamin

ingestion is associated with reduced cardiovascular disease, but the results of

randomised controlled trials are mainly disappointing. It has been suggested that natural

antioxidants may be effective only in selected subgroups of patients with high levels of

oxidative stress or depletion of natural antioxidant defence systems.17 The favourable

effects shown by some studies relating antioxidant dietary intake and cardiovascular

disease, may have been exerted by other chemicals present in foods. Flavonoids are the

ideal candidates, since they are plentiful in foods containing antioxidant vitamins (i.e.

fruits and vegetables) and are potent antioxidants. Tea and wine, rich in flavonoids,

seem to have beneficial effects on multiple mechanisms involved in atherosclerosis.

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Table 1.1. Epidemiological studies on antioxidants

Disease Antioxidant

Cancer

Gastric cancer Vit E, β-carotene, Selenium

Lung cancer in smokers Vit E, β-carotene both together

Prostate cancer Vit E

Lung cancer in workers exposed to

asbestos

Vit A + β-carotene

Lung cancer α,β-carotene, lutein, lycopene and β-crypto-xanthine

in diet for 10 years

Cardiovascular diseases

Myocardial infarction Aspirin

Coronary heart disease Vit E

Atherosclerosis Vit E

Stroke and myocardial infarction N-3 PUFA , Vit E And both together

Cardiovascular disease Catechin, Quercetin

Coronary heart disease Vit C

Hypertension Vit C

Heart failure Carvedilol (25 mg bid) Metoprolol (50 mg bid) for 4,

8, 12 weeks.

Neurodegenerative diseases

Parkinson’s disease Vit E. (2000 UI/day), Deprenyl (10 mg/day)and in

combination for 14 months

Alzheimer’s disease Selegiline (10mg/day), Vit E (2000 UI/day) and in

combination for 2 yrs

Others

Diabetes/hyperglycemia Vit C (24 mg/min) intra arterially for 10 min

Type 2 diabetes Vit E

Renal dysfunction Acetylcysteine (600 mg bid) i.v.

Subarachnoid hemorrhage in mice Transgenic Cu-Zn SOD (22.7 U/mg protein) as

compared to 7.9 in non transgenic mice

Pre-eclampsia Vit C (1000 mg) + Vit E (400 mg) during pregnancy

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1.4. Uses of antioxidants in technology

Food preservatives

Antioxidants are used as food additives to help guard against food deterioration.

Exposure to oxygen and sunlight are the two main factors in the oxidation of food, so

food is preserved by keeping in the dark and sealing it in containers or even coating it

in wax, as with cucumbers. However, as oxygen is also important for plant respiration,

storing plant materials in anaerobic conditions produces unpleasant flavors and

unappealing colors.18 Consequently, packaging of fresh fruits and vegetables contains

an ~8% oxygen atmosphere. Antioxidants are an especially important class of

preservatives as, unlike bacterial or fungal spoilage, oxidation reactions still occur

relatively rapidly in frozen or refrigerated food.19 These preservatives include natural

antioxidants such as ascorbic acid and tocopherols, as well as synthetic antioxidants

such as PG, TBHQ, BHA and BHT.20

The most common molecules attacked by oxidation are unsaturated fats;

oxidation causes them to turn rancid.21, 22 Antioxidant preservatives are also added to

fat-based cosmetics such as lipstick and moisturizers to prevent rancidity.

Industrial uses

Antioxidants are frequently added to industrial products. A common use is as

stabilizers in fuels and lubricants to prevent oxidation, and in gasoline’s to prevent the

polymerization that leads to the formation of engine-fouling residues.23

1.5. Heterocyclic compounds

Of the more than 20 million chemical compounds currently registered about one

half contains heterocyclic systems. Heterocycles are important, not only because of

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their abundance, but due to their chemical, biological and technical significance.

Heterocycles count among their number many natural products, such as vitamins,

products of technical importance (corrosion, inhibitors, anti aging drugs, sensitizers,

stabilizing agents etc.)

The study of heterocyclic compounds is an evergreen field in the branch of

organic and medicinal chemistry this always attracts the attention of scientists working

not only in the area of natural products but also synthetic organic, bio-organic and

medicinal chemistry. Many useful drugs have emerged from the successful

investigations carried out in this branch. More over spectacular advances have been

made to furtherance the knowledge of relationship between chemical structure and

biological activity this tendency is in fact reflected by the voluminous data available in

literature on hetero cyclic chemistry. Thus the successful application in various fields

ensures a limitless scope for the development of structurally novel compounds with a

wide range of physicochemical and biological properties.

Heterocyclic ring systems have emerged as powerful scaffolds for many

biological evaluations. 24 Heterocyclic compounds provide scaffolds on which

pharmacophores can arrange to yield potent and selective drugs. 25 Heterocyclic

compounds carrying piperidine skeleton are attractive targets of organic synthesis

owing to their pharmacological activity and their wide occurrence in nature.

1.6. Oximes

Oximes (R1R2C=N-OH) are a major class of hydroxylamines (R1R2NOH),

where R1 represents an organic side chain (i.e., alkyl, cycloalkyl or an aromatic moiety)

and R2 is either hydrogen, forming aldoxime, or another aromatic group, forming

ketoxime.26 The oxime functional groups can be transformed into such important

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groups as carbonyl, amino, nitro and cyano functions and can also serve as a convenient

protective group in synthetic chemistry applications.27

1.7. Biological significance of oxime derivatives

Oximes and oxime ethers (RRC=N-OR) have important pharmaceutical and

synthetic applications. The oxime (and oxime ether) functional group is incorporated

into many organic medicinal agents, including some antibiotics, for example,

gemifloxacin mesylate, pralidoxime chloride and obidoxime chloride (Figure 1.4) are

used in the treatment of poisoning by organophosphate insecticides like malathion and

diazinon.

Figure 1.4. Structure of some antibiotics

Oximes are generally used as chemical building blocks for the synthesis of

agrochemicals and pharmaceuticals.28 Oximes exhibited a protective role during in-

vitro Cu2+-induced oxidation of LDL and serum.29 Flavanone oxime derivatives

(ethers) have been shown to modulate the growth of Yoshida Sarcoma cells in vivo and

to induce apoptosis, but compared to anticancer drugs (doxorubicin, aclarubicin and

mitoxantrone), flavanone oximes displayed cytotoxicity at considerably higher

concentrations.30

In inorganic chemistry, oximes act as a versatile ligand. The stability of oxime

complexes with various metals has been shown to result in promising compounds with

antitumor activity, such as cis and trans platinum complexes 31 and homo and

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heteronuclear Cu(II) and Mn(II) oxime complexes.32 A complex of technetium with

hexamethylpropyleneamine oxime was also used to monitor photodynamic therapy of

prostate tumors.33 Some oxime complexes has reported remarkable DNA binding,

antioxidant, antimicrobial activities.34

Recently, pyrazole oxime derivatives have attracted considerable attention in

chemical and medicinal research because of their diverse bioactivities. They are widely

used as fungicide, insecticide, acaricide, and antitumor agents.35-39 Fenpyroximate is

currently used for the control of mites on various crops.40, 41 However, several field

populations of T. urticae have already develops high levels of Fenpyroximate resistance

despite its short-term use and chemists have begun to study cross-resistance patterns of

Fenpyroximate-resistant T. urticae, structural modification of Fenpyroximate and

corresponding effects on the biological activities.42-45

There are a large number of pharmaceuticals containing an oximino group

attached to a variable structure, frequently a heterocyclic one.46 Some oxime

derivatives present a fungitoxic and herbicide effect, 47,48 or act as growth regulators for

plants. Some α-oximinoketones are known to be important intermediates for the

synthesis of aminoacids,49 nitrosopyrazoles,50 2-vinylimidazoles,51 and so on.

Oxime moiety offers a convenient structural moiety for probing an element of

the pharmacophore which had proven to be of considerable importance in the

bezimidazole-isatin oximes and profiled as inhibitors of respiratory syncytial virus

(RSV) replication in cell culture.52 3-keto-clarithromycin 9-O-(3- aryl-E-2-propenyl)

oxime derivatives exhibited improved antibacterial activities against erythromycin-

susceptible Staphylococcus aureus and Streptococcus pneumonia and greatly enhanced

activities against the resistant strains encoded by erm and mef genes, as compared to

clarithromycin and azithromycin. 53

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One of the structurally distinct class of antiepileptic drugs are (arylalkyl)

imidazoles, Nafimidone and denzimole. Structure–activity relationship studies show

that anticonvulsant properties of this group are associated with the presence of a small

oxygen functional group (such as carbonyl, ethylene dioxy, methoxy, acyloxy and

hydroxy substituents) in the alkylene bridge in addition to imidazole ring 54 and

lipophilic aryl portion facilitating penetration of the blood–brain barrier. The

introduction of oxime and oxime ether groups to the alkylene bridge of (arylalkyl)

imidazoles as a small oxygen functional group led to oxime and some oxime ether

derivatives of nafimidone was reported as potential anticonvulsant compounds.55

A series of bis-pyridinium oximes connected by xylene linker were evaluated

for their in-vitro reactivation potential against acetylcholinesterase (AChE) inhibited by

nerve agent, sarin.56 A series of novel (Z )- and (E )-2-imidazolo-/2-triazolo-methyl

tetrahydronaphthyl oxime ethers conformationally rigid analogues of oxiconazole

(Figure 1.5.) has been reported as potent antimicrobial agents.57

Figure 1.5. Structures of oxime derived pharmacologically active drug oxiconazole

A new series of bis-pyridinium oximes bearing (E ) and (Z )-but-2-ene linkers,

which showed promising reactivation ability against chlorpyriphos and paraxon

inhibited AChE.58 Such bis-pyridinium oximes (Figure 1.6.) have shown promising

reactivation profile against organo phosphorus (OP)-inhibited AChE. In the past,

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reactive oxime moiety was incorporated into the variety of micelle forming molecules,

resulting in significant enhancement of hydrolysis of OP compounds. 59

Figure 1.6. Bis-pyridinium oximes used as reactivators of OP-inhibited AChE

Quinolinone oxime sulffmic acid compounds are novel diuretics, clearly

different from conventional diuretics in chemical structure but possessing functional

groups essential to interact with the cotransporter.60

N

NOSO3K

O

CH3Cl

Figure 1.7. Structure of potent diuretic quinolinone oxime sulffmic acid

Structure of potent diuretic quinolinone oxime sulffmic acid are shown in Figure 1.7.

Oximes as anticancer agents

Besides to this oxime-containing molecules caught attention, as they appear to

be amenable to biotransformation and conjugations with organic and inorganic

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molecules. The properties of these classes of compounds have been recently exploited

with the aim to design and develop novel therapeutic agents that can display acyl group

transfer capabilities and serve for the evaluation of novel candidate drugs for the

treatment of various diseases. For example, furan oximes were found to inhibit DNA,

RNA and protein synthesis in lipoid leukemia cells.61 Derivatives of quinoline oximes

were also shown to possess antitumor activity62 and glucosinolates were suggested as

cancer preventive agents.

There are several approaches for the therapy of breast cancer but the most

effective way to treat hormone-dependent breast cancer is to inhibit the key enzyme in

oestrogen biosynthesis i.e. aromatase cytochrome P450 (P450 arom) by making use of

aromatase inhibitors. Among steroidal aromatase inhibitors, (Figure 1.8) 4-hydroxy

androstenedione (4-OHA, Formestane) (a) has been approved for clinical use in the

treatment of breast cancer in several countries. Oximino derivatives, 6-hydroxiimino-4-

en-3- ones (b) and (c) also show a high affinity for human placental aromatase and

function as competitive inhibitors of this enzyme.63

Figure 1.8. Structures of steroidal aromatase inhibitors (a) Formestane, (b, c) 6-

Hydroxiimino-4-en-3-ones

Anthracenone-based oxime ethers and esters (Figure 1.9.) are considered to

contribute to the development of novel antiproliferative drugs, based on tubulin

interaction. Several investigated compounds displayed strong antiproliferative activity

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against K562 leukemia cells and proved to be strong inhibitors of tubulin

polymerization.64

Figure 1.9. Structure of Anthracenone-based oxime ether and ester derived

Antiproliferative agents

Oxime chemistry

The synthesis of conjugate vaccines can be a complex and expensive process

involving the preparation of the individual components, the chemical activation of the

polysaccharide and protein, a conjugation step and the purification of the conjugate

from its components.65

Applications of oxime chemistry are described for the efficient bioconjugation of

proteins and polysaccharides for the preparation of conjugate vaccines. In this process,

aldehyde or ketone carbonyls are reacted with the highly nucleophilic aminooxy (AO)

group to form oximes. Either the protein or the carbohydrate moiety can be

functionalized with aminooxy groups. In contrast to reductive amination, which yields

an unstable Schiff base, aminooxy groups rapidly condense with aldehydes or ketones

to form stable oximes, as indicated in Eq.(1).

(1) R1CHO + NH2OR2→ R CHNOR2

Coupling using oxime chemistry is efficient and can be effected over a wide pH

range. Furthermore, by limiting the number of cross-links between the protein and

Chapter-1

23

polysaccharide, control can be exerted over the degree of cross-linking. The approaches

described are compatible and complementary to a number of chemistries currently used

in conjugate vaccine synthesis. Oxime chemistry can be used to both simplify the

synthesis of and increase yields of conjugate vaccines. Mice immunized with

pneumococcal type 14 conjugates that were made using oxime chemistry mounted

significant anti-polysaccharide immune responses. The primary immune response could

be boosted, indicating that the polysaccharide conjugate had characteristics of a T cell

dependent antigen.

Oximes as antioxidant

The antiradical and antioxidant activities of four biologically active N,N-

diethyloaminoethyl ethers of flavanone oximes were investigated and these compounds

were shown to be promising antioxidants and radioprotectors comparable to rutin

activities, rendering them useful under oxidative stress conditions.66 Because of the

important potential uses of oximes, especially of flavonone oximes, The antioxidant

properties of naringenin oxime has been identified by using the cupric ion reducing

antioxidant capacity (CUPRAC) method. 67

Naringenin (4,5,7-trihydroxyflavanone) is a member of the flavonoid family

(flavanone) that is considered to have various bioactivities on human health as

antioxidant, ROS scavenger, monoamine oxidase inhibitor, and especially a most

investigated cancer preventive agent . Naringenin is a naturally available antioxidant

that can be used as starting material for the synthesis of other novel antioxidants with

enhanced antioxidant activity. Li et al. synthesized a new naringenin -derivatized

compound, naringenin - 2-hydroxy benzoyl hydrazone, and this compound was found

to possess higher antioxidant activity than simple naringenin.

Chapter-1

24

Because of the important potential uses of oximes, especially of flavonone

oximes, naringenin was derivatized as naringenin oxime (Figure 1.10) and their

antioxidant activity was evaluated. 66, 69

Figure 1.10. Structure of naringenin oxime

Natural occurrence of oxime in plants

Sponges of the family Verongidae provide a series of closely related

compounds which may be considered as metabolites of 3, 5-dibromotyrosine, including

aerothionin (1), homoaerothionin and the nitrile aeroplysinin (2).The spiro system in

(1) and (2) could arise in various ways, including nucleophilic attack by an oxime

function on an arene oxide as shown in Figure 1.11. It has been speculated that the

oxime (i) could be also a likely precursor of the nitrile aeroplysinin-1. There was a

report on the isolation of 4-hydroxyphenylpyruvic acid oxime (ii) (oximinopyruvic

acid) from a marine sponge, Hymeniacidon sanguinea.70

Figure 1.11. Structure of 4-hydroxyphenylpyruvic acid oxime isolated from a marine

sponge Hymeniacidon sanguinea.

Chapter-1

25

Since the discovery that indolyl-3-acetonitrile (IAN) occurs naturally and shows

auxin activity in certain plant species. Other oximes and particularly a-keto acid

oximes, have, however, been isolated from natural sources71 and the formation of

indolyl-3-acetaldoxime from indolyl-3-acetaldehyde and hydroxylamine is a tenable

supposition, since indirect evidence for the occurrence of both these substances in

plants is available.72

There was report on the chemical and chromatographic properties of synthetic

indolyl-3-pyruvic acid oxime and have found them to be very similar to those of the

IAN precursor. A new hypothesis has been there for the biosynthesis of IAN with

indolyl-3-pyruvic acid oxime as the key intermediate (Figure 1.12.).

Figure 1.12. Biosynthesis of IAN with indolyl-3-pyruvic acid oxime as the key

intermediate.

1.8. Synthesis of oximes

Synthesis of oximes is an important reaction in organic chemistry, because these

versatile oximes are used for protection, purification and characterization of carbonyl

compounds. Nitriles, amides via Beckmann rearrangement, nitro compounds, nitrones,

amines, and azaheterocycles can be synthesised from oximes. They also find

applications for selective α-activation.

Numerous functional group transformations of oximes make them very

important in synthetic organic chemistry. Among other synthesis applications, these

compounds were successfully transformed into amides73, amines74, hydroxylamines75,

Chapter-1

26

hydroxylamine O-ethers,76 nitroalkanes, 77 1,3-oxazoles, -thiazoles, and –diazoles,78

etc. Therefore, synthetic organic chemists are interested in a facilitation of oxime

synthesis. Although alternative methods exist,79 reaction of carbonyl compounds with

hydroxylamine hydrochloride remains still the most important route. The classical

method involves refluxing of an alcoholic solution of these reactants in the presence of

sodium acetate or hydroxide.80 Many improvements of this methodology have been

described. Thus, treatment of ketones with hydroxylamine hydrochloride in the

presence of an ion-exchange resin (Amberlyst A-21) as the catalyst in ethanol gave

oximes in high yields at room temperature and with a simple work-up procedure.81

a) From ethyl acetoacetate

Scheme-1.1.

To the solution of ethyl acetoacetate and glacial acetic acid 95 per cent sodium

nitrite in water was added. The mixture is stirred for one and half hour longer and then

allowed to stand for four hours to get desired oxime.82

b) From aldehyde

Scheme-1.2.

In the presence of zinc oxide and without any additional organic solvents,

Beckmann rearrangement of several ketones and aldehydes were performed in good

yields.83

Chapter-1

27

c) From ketone

Scheme-1.3.

The synthesis of oximes from ketones involves a reaction with hydroxylamine

hydrochloride and sodium acetate/sodium sulphate resulting oximes are isolated in

good yields and excellent purity. 84

Scheme-1.4.

d) From allenes

Scheme-1.5.

The reaction of monosubstituted allenes with aqueous hydroxylamine gives

oximes in good yields.85

e) From nitro alkanes

Scheme-1.6.

A convenient, environmentally friendly method for the synthesis of optically

active aldoximes and nitriles starting from chiral nitroalkanes was developed.86

Chapter-1

28

1.9. Reactions of oxime derivatives

The Beckman rearrangement is well known as the most characteristic reaction

of oxime derivatives and using this reaction, amides are produced when oximes or their

derivatives react with either acid or base. This rearrangement reaction is used not only

for synthesis of amides but also for synthesis of various heterocyclic compounds using

N-alkyl nitrilium ion intermediates. Thus, substitution on the nitrogen atom of the

oxime can be easily achieved via the Beckman rearrangement (Figure 1.13.), oxime

derivatives can be widely used as electrophilic amination reagents for synthesis of

amines and heterocyclic compounds containing nitrogen atoms.

Figure 1.13. Reactions of oximes

1.9.1 Substitution on oxime nitrogen atoms by SN2 type nucleophilic reaction

It is possible to catalytically induce a Beckmann rearrangement when n-

Bu4NReO4 and sulphonic acid were reacted with different oximes, resulting in the

generation of various perrhenic acid esters of oximes. Indeed, the Beckmann

rearrangement occurred and amides were produced when oximes were reacted with n-

Bu4NReO4 and trifluoromethane sulfonic acid (CF3SO3H) in a highly polar solvent

such as nitromethane87 (Scheme-1.7.).

Chapter-1

29

Scheme-1.7.

On the other hand, there are several reports in which N-N or N-S bond

formation took place at the nitrogen atom of oxime. For instance, it has been reported

that when an oxime having pyridyl group was reacted with tosyl chloride and pyridine

(Scheme-1.8.), a cyclic compound was produced from anti-isomer of the oxime, while

no cyclization occurred when the syn-isomer was used.88

Scheme-1.8.

1.9.1.1 Synthesis of cyclic imines using nucleophilic substitution reaction of

oximes.

When (E)-O-methylsulfonyloximes of ketones having active methylene

moieties at the γ and δ positions, were treated with DBU, an intramolecular

nucleophilic substitution took place, resulting in the quantitative production of five and

six membered cyclic imines (Scheme-1.9.). However, when the corresponding Z

isomers were used, no cyclic compounds were obtained at all. Moreover, when reaction

conditions were set for being more restricted, various reaction products such as Neber

reaction products were yielded.

Chapter-1

30

Scheme-1.9.

1.9.1.2 Alkylation of oxime derivatives by grignard reagents: synthesis of the

primary amines.

Nucleophilic species can attack on the oxime nitrogen atoms indicates that N-

alkylation can be undertaken by organometallic compounds using oxime derivatives

that rarely undergo the Beckmann Rearrangement. A report, in which a similar attempt

was made using O-tolylsulfonyl oxime derived from tetraphenylcyclopentadienone as

described below. However, it was reported that the reaction mechanism was through

addition/elimination but not substitution reactions (Scheme-1.10.) In this method, large

excess amounts of Grignard reagents were required to make the reaction take place.

Moreover, mono N-alkylation could dominantly occur only when an aromatic Grignard

reagent was used, while dialkylated compound was a major reaction product in cases

involving an alkyl Grignard reagent. This is because N-alkylimines formed by

monoalkylation are far more easily susceptible to addition reactions than O-

methylsulfonyl oxime.

Scheme-1.10.

Chapter-1

31

1.9.2. Radical cyclization reaction of oximes by one electron reduction

1.9.2.1 Synthesis of 8-quinolinol derivatives.

In the nucleophilic substitution reactions described above, it was considered to

be important to maintain a good balance between nucleophiles and leaving groups on

the oxime N atoms. Therefore, we attempted substitution using oxime derivatives

possessing various leaving groups. During this process, we found that 8-quinolinol and

its tetrahydro derivative were formed when m-hydroxyphenethyl ketone O-2,4-

dinitrophenyloxime was treated with sodium hydride (NaH). However, it was also

found that no 6-quinolinol, one of the position isomers, was produced at all. Moreover,

it was also demonstrated that both E- and Z-isomers of O-2,4-dinitrophenyloximes

exerted a similar reactivity (Scheme-1.11.).89

Scheme-1.11.

1.9.2.3 Catalytic radical cyclization reaction of oxime derivatives. Oxime was treated with acetic acid in the presence of 1,4-cyclohexadiene to

yield an acetoxy substituted dihydropyrrole as a product of the nucleophilic

substitution reaction, and its hydrogenated compound were produced (Scheme-1.12.).90

Scheme-1.12.

Chapter-1

32

1.9.3. Oxidative addition reaction of oximes to palladium catalysts

Since, low valent transition metal complexes are good electron donors, it was

expected that alkylideneamino metal complexes can be formed when O-substituted

oximes are oxidatively added to low valent transition metal complexes. In fact, when

benzophenone O-mesyloxime was reacted with an equimolar amount of Pd(PPh3)4,

followed by addition of water to the reaction mixture, imine was quantitatively

obtained (Scheme-1.13.). This result showed that an alkylideneaminopalladium

complex was formed as an intermediate.91

Scheme-1.13.

1.10. Scope of present investigation

Free radicals are responsible for causing a large number of diseases including

cancer, 92 cardiovascular disease,93 neural disorders,94 alzheimer’s disease,95 mild

cognitive impairment,96 Parkinson’s disease,97 alcohol induced liver disease,98

ulcerative colitis, 99 aging 100 and atherosclerosis. 101 Protection against free radicals can

be enhanced by ample intake of dietary antioxidants. Antioxidants are of great benefit

in improving the quality of life by preventing or postponing the onset of degenerative

diseases. In addition, they have a potential for substantial savings in the cost of health

care delivery. It is reasonable to assume that compounds functionalized with groups

endowed with potential antioxidant properties could be useful as new drugs for

chemoprevention and chemotherapy. There is, however, a growing consensus among

scientists about the synthetic antioxidants.

Chapter-1

33

In connection to this oxime-containing molecules caught our attention, as they

appear to be amenable to biotransformation and conjugations with organic and

inorganic molecules. They have important pharmaceutical and synthetic applications.

Oxime moiety offers a convenient structural moiety for probing an element of the

pharmacophore which had proven to be of considerable importance in the medicinal

chemistry. The properties of these classes of compounds have been recently exploited

with the aim to design and develop novel therapeutic agents that can serve for the

evaluation of novel candidate drugs for the treatment of various diseases. For example,

furan oximes were found to inhibit DNA, RNA, and protein synthesis in lipoid

leukemia cells.102, 103 Naringenin oximes were also shown to possess antioxidant

activity.104

In-depth literature on oximes has induced us to synthesize several oxime

derivatives and compared their antioxidant activities with standard antioxidants

butylated hydroxyl anisole (BHA) and ascorbic acid (AA) by the following in vitro

methods.

1. 2,2' –diphenyl-1-picrylhydrazyl (DPPH) assay,

2. 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assay,

3. Ferric reducing antioxidant power (FRAP) assay

4. Cupric ion reducing antioxidant capacity (CUPRAC) assay

5. Inhibition of microsomal lipid peroxidation

The present investigations give the simple and convenient routes for the

synthesis of novel oxime derivatives. The synthesized compounds shows promise

regarding their applications as antioxidants. The results obtained were very interesting,

encouraging and it opens a new pathway for further investigation in this area. These

Chapter-1

34

aspects which promise us to have pharmaceutical, industrial and commercial

applications. The results of scientific pursuit carried out in this regard have been

presented in six chapters.

Chapter 1: Introduction to free radicals, antioxidants and brief overview of oximes

Chapter 2

Part-A: Synthesis and antioxidant activity of novel vanillin derived piperidin-4-one

oxime esters: preponderant role of the phenyl ester substituents on the piperidin-4-one

oxime core

Part-B: 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one oxime esters: synthesis

and a new insight into their antioxidant potential

Chapter 3: Synthesis of thiazole based substituted piperidinone oximes: profiling of

antioxidant potential

Chapter 4: Synthesis, antioxidant evaluation and preliminary structure activity

relationship (SAR) study of novel 5-substiuted cytosine oxime esters

Chapter 5: Synthesis of novel hesperetin oxime esters: a new discernment in to their

antioxidant potential

Additional work

Chapter 6: Synthesis and antioxidant evaluation of novel indole-3-acetic acid

analogues

Chapter-1

35

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